Sensitive detection of p53 gene mutations by a 'mutant enriched' PCR-SSCP technique.
For the rapid and sensitive detection of p53 'hot spot' mutations, we combined polymerase chain reaction based single-strand conformational polymorphism (PCR-SSCP) analysis with sequence specific-clamping by peptide nucleic acids (PNAs) in a one-step reaction tube protocol. For this purpose, we designed two PNA molecules comprising aa 246-250 of exon 7 and aa 270-275 of exon 8, respectively, to suppress the amplification of wild-type p53 allelic variants during PCR amplification. Using this method in a survey of 20 brush cytology samples from lung cancer patients, we were able to detect five p53 point mutations occurring in codons 248, 249 and 273 which could not be retrieved by conventional PCR-SSCP. Thus, allelic suppression by PNA molecules opens a way to largely improve the sensitivity of existing PCR-SSCP protocols (approximately 10-50-fold) and could be useful in the detection of 'hot spot' oncogene lesions in histological samples containing only a small number of cancer cells.
Behn
M
M
Zentrum für Innere Medizin, Abteilung Hämatologie/Onkologie, Philipps-Universität Marburg, Baldingerstrasse, D-35033 Marburg, Germany.
Schuermann
M
M
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005091:Exons; D016158:Genes, p53; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D009154:Mutation; D009696:Nucleic Acids; D010455:Peptides; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004273:DNA, Neoplasm; D009696:Nucleic Acids; D010455:Peptides